Protein glycosylation is a fundamental process in living organisms. Analysis of the frequency of glycosylation has predicted that more than half of all proteins in nature will eventually be identified as glycoproteins. Without these added carbohydrates, the function of many proteins is aberrant. Complex carbohydrates are involved in cellular communication via cell/cell contact, metastasis (the spread of cancer cells through the body), viral and bacterial adhesion, and binding of toxins to cells. Understanding the roles of carbohydrate biology is crucial to basic health research and to the pharmaceutical industry.
Although protein glycosylation is rare in Escherichia coli, it is a common phenomenon in other bacteria. Bacteria can tolerate the manipulation of their glycosylation systems and are therefore useful for glycoengineering. In this respect, the use of bacteria to produce O-glycosylated recombinant proteins is described in Faridmoayer, et al. ((2007) J. Bacteriol. 189(22)8088) and U.S. Pat. No. 6,872,398 ('398 patent). Specifically, the '398 patent teaches a multivalent vaccine against Gram-negative bacterial infections composed of heterologously glycosylated pili from Pseudomonas aeruginosa. To produce this vaccine, the '398 patent teaches the introduction into a Gram-negative bacterium, of a vector containing pilA, the pilin structural gene from P. aeruginosa, and pilO, the gene from P. aeruginosa coding for the protein responsible for the attachment of the O-antigen repeating unit to the pilin subunit. Once expressed, PiIO adds the O-antigen repeating unit of the host Gram-negative bacterium to the pilin protein PiIA. The O-glycosylated pilin is then purified from a culture of the transformed bacteria. However, PiIO is unable to transfer glycans to internal glycosylation sites in proteins to be glycosylated thereby limiting its use.
WO/1997/043405 also suggests the use of bacteria to express human UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase (GalNAc-T3) and the production of glycosylated polypeptides having particular enzymatic, immunogenic, or other biological or physical properties. Similarly, U.S. Pat. No. 6,916,649 suggests the use of bacteria to express human UDP-galactose: β-N-acetylglucosamine β-1,4-galactosyltransferase (GalNAc-T2). U.S. Pat. Nos. 7,045,337 and 7,378,263 also generally refer to the expression of an N-acetyl-galactosamimidase, a transglycosylase, or a serine-glycosylhydrolase to glycosylate amino acids in bacteria. Furthermore, enzymatic synthesis for the preparation of GalNAc-α-linked compounds using glycosyltransferases and glycosidases obtained from natural sources or from recombinant cells is suggested in U.S. Pat. No. 5,882,902.
Likewise, WO/2008/128230 teaches the production of reference glycoproteins such as antibodies, fusion proteins and hormones having defined glycan structures. This reference teaches the use of E. coli and expression of enzymes that cleave polysaccharides such as degrading enzymes, enzymes that add monosaccharides to a glycan structure, enzymes that remove a component of a monosaccharide, enzymes that add a component to a monosaccharide and enzymes that convert a chemical unit into a different chemical unit.
While O-glycosylation of therapeutic proteins in prokaryotes has been suggested by co-expressing the therapeutic protein and a heterologous glycosyltransferase that transfers a sugar moiety to an amino acid acceptor on the therapeutic protein (US 2009/0311744), O-GalNAc-T has been suggested to form inactive inclusions when expressed in E. coli (Ramakrishnan, et al. (2007) Bioconjug. Chem. 18(6):1912-8). In this respect, improved methods for expression in prokaryotes are needed. Moreover, suitable donor substrates need to be provided to the above-described cells in order to synthesize glycoproteins.